1. Field of the Invention
The present invention relates to a chaperone expression plasmid. More particularly, the present invention relates to an operon comprising polynucleotides encoding each of chaperones DnaK, DnaJ and GrpE; an expression plasmid carrying the operon; a cotransformant prepared by introducing the expression plasmid into Escherichia coli (hereinafter simply referred to as "E. coli") together with an expression vector for a foreign protein; and a method for producing a foreign protein using the cotransformant.
2. Discussion of the Related Art
E. coli serves ideally as a host for production of heterologous proteins at low costs and high yields, because it can easily be grown to high densities and the studies on the host-vector systems have been most advanced and many high-expression vectors have been developed. E. coli host-vector systems are, therefore, most widely utilized as expression systems for heterologous genes.
However, many heterologous proteins, especially eukaryotic proteins, associate with each other in cytoplasm and form biologically inactive insoluble aggregates known as "inclusion bodies" when expressed at high levels in E. coli. There is an advantage in formation of an inclusion body in that it is made possible to protect the expressed protein against degradation by proteases in host cells and to easily separate the inclusion body by centrifugation from the cells. In order to obtain the desired biologically active protein, however, it is necessary for the inclusion body to be denatured and solubilized, followed by renaturation (refolding). This solubilization-renaturation process is performed on the basis of repeated trial and error for individual proteins, but often fails to achieve satisfactory recovery rates. In some cases, renaturation is not always possible. Also, not a few heterologous proteins are degraded by proteases in E. coli and fail to achieve high expression levels. There have not yet been found a well-established means for solving such problems of insolubilization and degradation of expression products. Attempts to mass-produce biologically active proteins in E. coli have not always been altogether successful. In order to solve this problem, coexpression of chaperones and the like has been known, and a number of reports have been made.
DnaK, DnaJ and GrpE are chaperones that cooperatively act in protein folding. It has been considered that the ATP bound to DnaK is first hydrolyzed upon DnaJ binding to an unfolded protein substrate, resulting in the formation of an unfolded protein-DnaJ-DnaK (ADP binding type) complex, and thereafter ADP/ATP exchange takes place by GrpE, resulting in the release of the protein substrate from the complex [Szabo, A. et al., Proc. Natl. Acad. Sci. USA 91, 10345-10349 (1994)].
The dnaK and dnaJ genes are located at the same operon on the E. coli chromosome, while the grpE gene is located at a site apart from the above operon. To date, there have been reported a method of coexpression of a desired protein with DnaK alone or with both DnaK and DnaJ [Blum, P. et al., BioTechnol. 10, 301-304 (1992); Perez--Perez, J. et al., Biochem. Biophys. Res. Comm. 210, 524-529 (1995)]; a method of coexpression of a desired protein and DnaJ alone (Japanese Patent Laid-Open No. Hei 8-308564); a method of expression of DnaK and DnaJ, and of GrpE from respectively different plasmids [Caspers, P. et al., Cell. Mol. Biol. 40, 635-644 (1994)]; and a method of independent expression of DnaK and DnaJ and of GrpE from the same plasmid using the same promoter [Stieger, M. and Caspers, P., Immunology Methods Manual, 39-44 (1997)]. However, these methods have the drawbacks described below.
Specifically, DnaK, DnaJ and GrpE, which act in cooperation with each other, are expected to be more effective when coexpressed, and it is very likely that their inherent chaperone function is not fully exhibited simply when DnaK alone or only DnaK and DnaJ are expressed. Also, in the method in which DnaK and DnaJ, and GrpE, are expressed from the respectively different plasmids, since it is difficult for a total of three plasmids, including the expression plasmid for the desired protein, to coexist in E. coli, the gene for GrpE and the gene for the desired protein are placed on a single plasmid, which in turn necessitates that the expression plasmids need to be constructed to adapt to individual desired proteins. Moreover, since the same promoter is used for expression of GrpE and the desired protein, there arises a defect in that the expression of the desired proteins cannot be increased to sufficient levels. Further, in the method in which DnaK and DnaJ, and GrpE, are independently expressed from the same plasmid using the same promoter, another problem arises in the plasmid stability because of the presence of two units of the same promoter.
It has been well known to use protease mutants of E. coli as hosts to reduce the degradation of foreign proteins in E. coli. For example, deletion mutants for Lon proteases are preferably used. In addition, there has been known a method using rpoH mutants to suppress Lon and Clp proteases, since the induction of their expression is controlled by .sigma..sup.32, encoded by the rpoH gene (Japanese Unexamined Patent Publication No. Sho 61-501307, WO 85/03949). Also, there has been known a method for stably expressing foreign proteins using double-mutants having mutations in the clpPX and lon genes (Japanese Patent Laid-Open No. Hei 8-140671).
It should be noted, however, that .sigma..sup.32 also controls the induction of expression of chaperones, such as DnaK, DnaJ, GrpE, GroEL and GroES. GroEL and GroES are essential for the growth of E. coli, and rpoH deletion mutants cannot grow at temperatures exceeding 20.degree. C. Therefore, missense mutations have conventionally been used for rpoH mutants (htpR mutants). It is desired, however, that the rpoH deletion mutants be used to more completely suppress the induction of expression of various proteases, such as Lon protease and Clp protease.
There have been reported a large number of successful cases of solubilization of foreign proteins that otherwise remain insolubilized in E. coli by coexpression of the foreign protein and GroEL and GroES. Examples thereof include, for instance, tyrosine kinase [Caspers, P. et al., Cell Mol. Biol. 40, 635-644 (1994); Amrein, K. E. et al., Proc. Natl. Acad. Sci. USA 92, 1048-1052 (1995)]; glutamate racemase [Ashiuchi, M. et al., J. Blochem. 117, 495-498 (1995)]; and dihydrofolate reductase [Dale, G. E. et al., Protein Eng. 7, 925-931 (1994)]. Other reported cases include improvement of solubility of human growth hormone by coexpression of DnaK [Blum, P. et al., Biotechnol. 10, 301-304 (1992)], transglutaminase solubilization by coexpression of DnaJ (Japanese Patent Laid-Open No. Hei 8-308564), and tyrosine kinase solubilization by coexpression of DnaK, DnaJ and GrpE [Caspers, P. et al., Cell Mol. Biol. 40, 635-644 (1994)]. It remains very difficult, however, to predict which foreign protein and which chaperone are to be coexpressed to what extent.